CN1243022C - Biological modifying and recombinant human growth hormone compound and preparing method thereof - Google Patents
Biological modifying and recombinant human growth hormone compound and preparing method thereof Download PDFInfo
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- CN1243022C CN1243022C CN 200310107990 CN200310107990A CN1243022C CN 1243022 C CN1243022 C CN 1243022C CN 200310107990 CN200310107990 CN 200310107990 CN 200310107990 A CN200310107990 A CN 200310107990A CN 1243022 C CN1243022 C CN 1243022C
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Abstract
The present invention relates to a recombinant human growth hormone compound modified by biology, which belongs to the technical field of peptide, biologic modifying substances of protein, and the preparation thereof. The recombinant human growth hormone with glutamine residue and polyethyleneglycol alkylamine PEG of an amino group donor substance or single methoxypolyethylene glycol mPEG react under the catalysis of transglutaminase (mTG), amido bonds are formed on amide in the gamma-position of the glutamine residue of the recombinant human growth hormone and an amino group in the uncle ammonia position of the amino group donor substance; therefore, the recombinant human growth hormone compound modified by biology is obtained, namely PEG-rhGH or mPEG-rhGH. The recombinant human growth hormone compound modified by biology has the advantages of high biological stability and long half-life in vivo, and is suitable for preparing long-effect injection type medicine or developing oral administration type medicine.
Description
Technical field
The present invention relates to a kind of bio-modification recombinant human somatropin mixture and preparation method thereof, belong to the technical field of peptide or proteinic bio-modification thing and preparation thereof.
Background technology
Human growth hormone (hGH) is by a kind of polypeptide hormone of being made up of 191 amino acid of people's Anterior pituitary excretory.People obtain recombinant human somatropin (rhGH) by genetic engineering technique.U.S. FDA in 1985 has been ratified rhGH and has been gone on the market as medicine, China also goes on the market as medicine in approval in 1998, and (Li Yuan edits " genetically engineered drug ", Chemical Industry Press, in December, 2002 first version P3-5), be mainly used in the spontaneous growth hormone deficiency dwarfism of treatment children, also can be used for treating burn, fracture, wound, hemorrhagic ulcer, Tener syndromes, tissue necrosis, muscular dystrophy, osteoporosis and obesity etc. clinically.The recombinant human somatropin is a kind of activated protein, be easy in vivo be caused biologically stable poor by proteasome degradation, recombinant human somatropin's molecular weight is less relatively simultaneously, be 21000 dalton, be easy to by glomerular filtration, thereby the transformation period short (having only about 30 minutes) in the body, need prolonged and repeated drug administration by injection, generally need 3 months to 2 years successive administrations, bring misery and inconvenience to patient.
Summary of the invention
First technical problem that the present invention will solve is to propose a kind of bio-modification recombinant human somatropin mixture, it is characterized in that, it is by recombinant human somatropin rhGH and amino donor substance polyethyleneglycol derivative---and polyalkylene glycol alkyl amine PEG or mono methoxy polyethylene glycol amine mPEG form, contain glutamine residue in recombinant human somatropin's the aminoacid sequence, amino donor substance contains amino, amino on uncle's ammonia position of the γ position acid amides of recombinant human somatropin's glutamine residue and amino donor substance links together by amido linkage, the mol ratio of recombinant human somatropin and amino donor substance is 1: 1, and molecular weight is not less than 24350 dalton.This mixture has the advantage of long half time in biologically stable height and the body.
Second technical problem that the present invention will solve provides the preparation method of bio-modification recombinant human somatropin mixture.The technical scheme that the present invention solves the problems of the technologies described above is to have recombinant human somatropin and the amino donor substance polyalkylene glycol alkyl amine PEG or the mono methoxy polyethylene glycol amine mPEG of glutamine residue, under the catalysis of trans-glutaminases mTG, react, amino on uncle's ammonia position of the γ-position acid amides of recombinant human somatropin's glutamine residue and amino donor substance forms amido linkage, thereby obtain bio-modification recombinant human somatropin mixture, i.e. PEG-rhGH or mPEG-rhGH.
Now describe technical scheme of the present invention in detail.
A kind of preparation method of bio-modification recombinant human somatropin mixture is characterized in that operation steps is as follows:
The first step, the 100mM that the recombinant human somatropin who takes by weighing 1 part of weight is dissolved in 1000 parts of weight contains in the Tris-HCl pH6.0 solution of 5mMEDTA, makes it dissolving;
Second step, the amino donor substance that in the first step solution, adds 0.8～16 part of weight, evenly mixed, the recombinant human somatropin to be finished and the mol ratio of amino donor substance are 1: 5～100 in the solution after mixed, described amino donor substance is a polyalkylene glycol alkyl amine, and molecular weight is 3350 dalton;
The 3rd step added trans-glutaminases mTG in the second step gained mixed solution, evenly mixed, making its final concentration is 0.1～1.0u/ml;
In the 4th step, the mixing solutions that contains mTG that previous step is handled places 4～55 ℃ of reaction 0.5～24h down;
In the 5th step, reaction is got 20 μ l reactants and is carried out SDS-PAGE electrophoretic examinations degree of modification after finishing;
The 6th step, get that the gel permeation chromatography post carries out separation and purification on the modification reaction mixture, the separation and purification condition is: the chromatography column Superdex 75 of 1 * 30cm, damping fluid are the 5mM PB pH7.4 that contains 0.15MNaCl, flow velocity is 0.5ml/min, and post is pressed and is 1.5Mpa;
The 7th step, the absorption peak solution of collection of biological modified and recombined human tethelin mixture;
The 8th step concentrated the mixture absorption peak solution of collecting, lyophilize, obtain 0.08～0.32mg pure, molecular weight is for being not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
Technical solution of the present invention is further characterized in that amino donor substance is a mono methoxy polyethylene glycol amine, and molecular weight is 5000 dalton, and consumption is 1.2～24 parts of weight.
Advantage of the present invention is by using trans-glutaminases mTG, under mild conditions, can will have the recombinant human somatropin of glutamy amido and have that the formation amido linkage forms the modification mixture between the amino polyethyleneglycol derivative.This biological enzyme modifying method has the reaction conditions gentleness, keeps recombinant human somatropin's biological activity easily; Because enzyme reaction needs certain sterically hindered, thereby the possibility height that reacts on recombinant human somatropin's specific position, so the by product kind is few, also is easy to control.This under the enzyme catalysis of mTG biological activity through recombinant human somatropin's mixture of polyethyleneglycol derivative bio-modification, because polyethyleneglycol derivative: PEG or mPEG form barrier on the recombinant human somatropin surface, make this recombinant human somatropin's mixture can not degraded, thereby the biologically stable of this recombinant human somatropin's mixture is improved by the body endoproteinase.Because the molecule quantitative change of modified recombinant human somatropin's mixture is big, is difficult for filtering renal glomerulus, and the interior transformation period of body of this recombinant human somatropin's mixture is prolonged greatly, thereby improved this recombinant human somatropin's bioavailability simultaneously.
Embodiment
All embodiment all operate according to above-mentioned preparation method.
One of preparation method of embodiment 1 bio-modification recombinant human somatropin mixture.In the present embodiment, the amino donor substance that is used for bio-modification is a polyalkylene glycol alkyl amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution that contains 5mM EDTA of 1g, makes it dissolving;
Second step added 0.8mg polyalkylene glycol alkyl amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and polyalkylene glycol alkyl amine mol ratio is 1: 5 in the solution after mixed;
The 3rd step added trans-glutaminases mTG in the second step gained miscellany, evenly mixed, making its final concentration is 0.1～1.0u/ml;
In the 4th step, the mixture that the 3rd step gained is contained mTG places 4～55 ℃ of reaction 0.5～24h down;
In the 5th step, reaction is got 20 μ l reactants and is carried out SDS-PAGE electrophoretic examinations degree of modification after finishing;
The 6th step, get that the gel permeation chromatography post carries out separation and purification on the modification reaction mixture, the separation and purification condition is: the chromatography column Superdex75 of 1 * 30cm, damping fluid are the 5mM PB pH7.4 that contains 0.15MNaCl, flow velocity is 0.5ml/min, and post is pressed and is 1.5Mpa;
The 7th step, the absorption peak solution of collection of biological modified and recombined human tethelin mixture;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.2～0.25mg pure, molecular weight is for being not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
The preparation method's of embodiment 2 bio-modification recombinant human somatropin mixtures two.In the present embodiment, the amino donor substance that is used for bio-modification is a polyalkylene glycol alkyl amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution that contains 5mM EDTA of 1g, makes it dissolving;
Second step added 8mg polyalkylene glycol alkyl amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and polyalkylene glycol alkyl amine mol ratio is 1: 50 in the solution after mixed;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.27～0.32mg pure, molecular weight is not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
The preparation method's of embodiment 3 bio-modification recombinant human somatropin mixtures three.In the present embodiment, the amino donor substance that is used for bio-modification is a polyalkylene glycol alkyl amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution that contains 5mM EDTA of 1g, makes it dissolving;
Second step added 16mg polyalkylene glycol alkyl amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and polyalkylene glycol alkyl amine mol ratio is 1: 100 in the solution after mixed;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.18～0.22mg pure, molecular weight is not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
The preparation method's of embodiment 4 bio-modification recombinant human somatropin mixtures four.In the present embodiment, the amino donor substance that is used for bio-modification is a mono methoxy polyethylene glycol amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution of the 5mM EDTA that contains 1g, makes it dissolving;
Second step added 1.2mg mono methoxy polyethylene glycol amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and mono methoxy polyethylene glycol amine mol ratio is 1: 5 in the solution after mixed;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.08～0.13mg pure, molecular weight is not less than 26000 daltonian bio-modification recombinant human somatropin mixture mPEG-rhGH.
The preparation method's of embodiment 5 bio-modification recombinant human somatropin mixtures five.In the present embodiment, the amino donor substance that is used for bio-modification is a mono methoxy polyethylene glycol amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution of the 5mM EDTA that contains 1g, makes it dissolving;
Second step added 12mg mono methoxy polyethylene glycol amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and mono methoxy polyethylene glycol amine mol ratio is 1: 50 in the solution after mixed;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.2～0.25mg pure, molecular weight is not less than 26000 daltonian bio-modification recombinant human somatropin mixture mPEG-rhGH.
The preparation method's of embodiment 6 bio-modification recombinant human somatropin mixtures six.In the present embodiment, the amino donor substance that is used for bio-modification is a mono methoxy polyethylene glycol amine.
The first step takes by weighing the 1.0mg recombinant human somatropin and is dissolved in the 100mM Tris-HCl pH6.0 solution that contains 5mM EDTA of 1g, makes it dissolving;
Second step added 24mg mono methoxy polyethylene glycol amine in the first step solution, evenly mixed, made that recombinant human somatropin's to be finished and mono methoxy polyethylene glycol amine mol ratio is 1: 100 in the solution after mixed;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The 8th step concentrated the modification mixture absorption peak solution of collecting, lyophilize, obtain 0.12～0.17mg pure, molecular weight is not less than 26000 daltonian bio-modification recombinant human somatropin mixture mPEG-rhGH.
Claims (3)
1, a kind of bio-modification recombinant human somatropin mixture, it is characterized in that, it is by recombinant human somatropin rhGH and amino donor substance polyethyleneglycol derivative---and polyalkylene glycol alkyl amine PEG or mono methoxy polyethylene glycol amine mPEG form, contain glutamine residue in recombinant human somatropin's the aminoacid sequence, amino donor substance contains amino, amino on uncle's ammonia position of the γ position acid amides of recombinant human somatropin's glutamine residue and amino donor substance links together by amido linkage, the mol ratio of recombinant human somatropin and amino donor substance is 1: 1, and molecular weight is not less than 24350 dalton.
2, the preparation method of the described bio-modification recombinant human somatropin of claim 1 mixture is characterized in that operation steps is as follows:
The first step, the 100mM that the recombinant human somatropin who takes by weighing 1 part of weight is dissolved in 1000 parts of weight contains in the Tris-HCl pH6.0 solution of 5mMEDTA, makes it dissolving;
Second step, the amino donor substance that in the first step solution, adds 0.8～16 part of weight, evenly mixed, the recombinant human somatropin to be finished and the mol ratio of amino donor substance are 1: 5～100 in the solution after mixed, described amino donor substance is a polyalkylene glycol alkyl amine, and molecular weight is 3350 dalton;
The 3rd step added trans-glutaminases mTG in the second step gained mixed solution, evenly mixed, making its final concentration is 0.1～1.0u/ml;
In the 4th step, the mixing solutions that contains mTG that previous step is handled places 4～55 ℃ of reaction 0.5～24h down;
In the 5th step, reaction is got 20 μ l reactants and is carried out SDS-PAGE electrophoretic examinations degree of modification after finishing;
The 6th step, get that the gel permeation chromatography post carries out separation and purification on the modification reaction mixture, the separation and purification condition is: chromatography column Superde * 75 of 1 * 30cm, damping fluid are the 5mM PB pH7.4 that contains 0.15MNaCl, flow velocity is 0.5ml/min, and post is pressed and is 1.5Mpa;
The 7th step, the absorption peak solution of collection of biological modified and recombined human tethelin mixture;
The 8th step concentrated the mixture absorption peak solution of collecting, lyophilize, obtain 0.08-0.32mg pure, molecular weight is for being not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
3, the preparation method of the described bio-modification recombinant human somatropin of claim 1 mixture is characterized in that operation steps is as follows:
The first step, the 100mM that the recombinant human somatropin who takes by weighing 1 part of weight is dissolved in 1000 parts of weight contains in the Tris-HCl pH6.0 solution of 5mMEDTA, makes it dissolving;
Second step, the amino donor substance that in the first step solution, adds 1.2～24 parts of weight, evenly mixed, the recombinant human somatropin to be finished and the mol ratio of amino donor substance are 1: 5～100 in the solution after mixed, described amino donor substance is a mono methoxy polyethylene glycol amine, and molecular weight is 5000 dalton;
The 3rd step added trans-glutaminases mTG in the second step gained mixed solution, evenly mixed, making its final concentration is 0.1～1.0u/ml;
In the 4th step, the mixing solutions that contains mTG that previous step is handled places 4～55 ℃ of reaction 0.5～24h down;
In the 5th step, reaction is got 20 μ l reactants and is carried out SDS-PAGE electrophoretic examinations degree of modification after finishing;
The 6th step, get that the gel permeation chromatography post carries out separation and purification on the modification reaction mixture, the separation and purification condition is: chromatography column Superde * 75 of 1 * 30cm, damping fluid are the 5mM PB pH7.4 that contains 0.15MNaCl, flow velocity is 0.5ml/min, and post is pressed and is 1.5Mpa;
The 7th step, the absorption peak solution of collection of biological modified and recombined human tethelin mixture;
The 8th step concentrated the mixture absorption peak solution of collecting, lyophilize, obtain 0.08-0.32mg pure, molecular weight is for being not less than 24350 daltonian bio-modification recombinant human somatropin mixture PEG-rhGH.
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Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1893239A2 (en) * | 2005-06-15 | 2008-03-05 | Novo Nordisk Health Care AG | Transglutaminase mediated conjugation of growth hormone |
| US20090117640A1 (en) * | 2005-08-18 | 2009-05-07 | Novo Nordisk Health Care Ag | Transglutaminase Variants with Improved Specificity |
| JP2009506096A (en) | 2005-08-30 | 2009-02-12 | ノボ ノルディスク ヘルス ケア アーゲー | Liquid preparation of pegylated growth hormone |
| US10351615B2 (en) | 2006-02-03 | 2019-07-16 | Opko Biologics Ltd. | Methods of treatment with long-acting growth hormone |
| US8048849B2 (en) | 2006-02-03 | 2011-11-01 | Modigene, Inc. | Long-acting polypeptides and methods of producing same |
| US10221228B2 (en) | 2006-02-03 | 2019-03-05 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US9249407B2 (en) | 2006-02-03 | 2016-02-02 | Opko Biologics Ltd. | Long-acting coagulation factors and methods of producing same |
| US8450269B2 (en) * | 2006-02-03 | 2013-05-28 | Prolor Biotech Ltd. | Long-acting growth hormone and methods of producing same |
| US9458444B2 (en) | 2006-02-03 | 2016-10-04 | Opko Biologics Ltd. | Long-acting coagulation factors and methods of producing same |
| US20140113860A1 (en) | 2006-02-03 | 2014-04-24 | Prolor Biotech Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US8946155B2 (en) | 2006-02-03 | 2015-02-03 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| US20150038413A1 (en) | 2006-02-03 | 2015-02-05 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
| CN101495155A (en) | 2006-07-07 | 2009-07-29 | 诺沃-诺迪斯克保健股份有限公司 | New protein conjugates and methods for their preparation |
| JP2010500886A (en) * | 2006-08-18 | 2010-01-14 | ノボ ノルディスク ヘルス ケア アーゲー | Transglutaminase variants with improved specificity |
| WO2008102008A1 (en) * | 2007-02-22 | 2008-08-28 | Novo Nordisk Health Care Ag | Transglutaminase variants with improved specificity |
| US9663778B2 (en) | 2009-07-09 | 2017-05-30 | OPKO Biologies Ltd. | Long-acting coagulation factors and methods of producing same |
| US20150158926A1 (en) | 2013-10-21 | 2015-06-11 | Opko Biologics, Ltd. | Long-acting polypeptides and methods of producing and administering same |
| CN108289851B (en) | 2015-06-19 | 2021-06-01 | Opko生物科学有限公司 | Long-acting coagulation factor and production method thereof |
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